A number of methods for culturing mammalian cells of different tissue origins have been reported. However, many of these cells are difficult to grow in vitro and, when grown, are not morphologically similar to in vivo tissue.
Green and Rheinwald (U S. Pat. No 4.016,036, 1977) describe a procedure for serially cultivating keratinocyte cells grown in the presence of mitotically inhibited fibroblasts. Without a second cell type (e.g.. 3T3 fibroblasts), the keratinocyte cultures were neither uniform nor differentiated. A major disadvantage of using tissue produced by this method for in vitro toxicology and other studies is the presence of fibroblasts. Although the fibroblasts are mitotically inhibited, they are still metabolically active. As such, the metabolic activity and/or cellular components of the fibroblasts interfere with assays for cells under study.
Woodley and co-workers (Joint Meeting of Amer. Chem. Soc. Cell. Biol. and Amer. Soc. Biochem. Mol. Biol., Abstract 4536, page 798a, Jan. 29-Feb. 2, 1989. San Francisco, Calif.) describe a method for growing keratinocyte cells on collagen without the use of a second cell type. A medium containing epidermal growth factor and bovine pituitary extract was employed to facilitate cell growth. Epidermal tissue grown by this method, however, cannot be raised to the air/liquid interface since there is no means for providing nutrition to the tissue.
A method for growing keratinocyte cells at the air/liquid interface has been described by Bernstam, I. L. et al., In Vitro Cell Dev. Biol. 22:695-704 (1986). Cells grown on a collagen substrate produced confluent monolayers; however, non-uniform areas of stratification were observed.
Thus, it would be desirable to produce a tissue which is morphologically similar to its in vivo conterpart for in vitro toxicology and other studies (for example, transepidermal drug transport).